Mutated proteins encoded by a lentivirus mutated env gene, peptide fragments and expression vectors

ABSTRACT

Mutated proteins coded by a mutated env gene of a lentivirus, particularly FIV, HIV or CAEV, peptide fragments contained in said mutated proteins and expression vectors expressing said mutaded proteins as well as the applications thereof are described. The peptide fragments are contained in the principal immunodominant domain (PID) of the transmembrane protein (TM) of lentiviruses and particularly of feline immunodeficiency virus (FIV), human immounodeficiency virus (HIV-1 and HIV-2), arthritis virus and caprine encephalitis virus (CAEV), and are capable of modifying the immunogenic properties of the PID domain and of the Env protein of the lentiviruses containing them. Such fragments have one of the following sequences: SEQ ID No1: CNQNQWLCK, SEQ ID No2: CNQNQFLCK, SEQ ID No3: CNQNQLWCK, SEQ ID No4: CNQNQPFCK, SEQ ID No5: CEHQHFFCK, SEQ ID No6: CSMGTFFCK, SEQ ID No7: CLTDSFFCK, SEQ ID No8: CELKNFFCK, SEQ ID No9: CRFAAFFCK, SEQ ID No10: ELGCGSKLICKIP, SEQ ID No11: IWGCNQNQFFCTTAVPWN, SEQ ID No12: IWGVAFRQVCTTAVPW.

This application is the national stage of PCT/FR96/00449, filed Mar. 26,1996.

The present invention relates to mutated proteins encoded by a mutatedenv gene of a lentivirus, and in particular FIV, HIV or CAEV, to peptidefragments included in the said mutated proteins, to expression vectorsexpressing the said mutated proteins and to their applications.

Feline immunodeficiency is due to a lentivirus, the felineimmunodeficiency virus (FIV), which has a genetic structure similar tothat of the lentiviruses of primates (HIV and SIV).

A number of fragments have been selected and have allowed thedevelopment of sensitive and specific tests for the detection ofseropositive animals, as described in European Patent Applications No.0,564,477 of Nov. 20, 1991, No. 0,577,458 of Jun. 16, 1993, and FrenchPatent Application No. 94 07062 of Jun. 9, 1994, in the name of theapplicant, and are derived, for the majority, from the Env protein ofFIV, comprising 854 amino acids, whose sequence is described in EuropeanApplication 0,577,458, which provides, after cleavage, 2 glycoproteinfragments called SU (surface glycoprotein) and TM (transmembraneglycoprotein).

In particular, the TM protein includes several fragments of interest,namely:

a fragment including a segment of 51 amino acids, called TM1, whichcorresponds to positions 595-647 of the Env protein of FIV,

a fragment including a segment of 31 amino acids, called TM2, whichcorresponds to positions 681-711 of the Env protein of FIV, the saidfragment contains an epitope including the sequence: Cys⁶⁹⁷--Asn--Gln--Asn--Gln--Phe--Phe--Cys--Lys⁷⁰⁵ (peptide called P237),

a fragment including a segment of 45 amino acids, called TM3, whichcorresponds to positions 744-788 of the Env protein of FIV, and

a fragment including a segment of 29 amino acids, called TM4, whichcorresponds to positions 826-854 of the Env protein of FIV.

Whereas in the field of detection, there are now available a range ofreagents for the detection of FIV, in the field of immunoprotection, ithas appeared that the principal immunodominant domain (PID) of the FIVenvelope protein, comprising the abovementioned peptide P237, can causethe formation of antibodies which facilitate viral infection, whoseaction has a deleterious effect opposite that of the protectiveantibodies generated by vaccination with the Env protein (J. R. MASCOLAet al., AIDS Research & Human Retroviruses, 1993, 9, 12, 1175-1184).

In addition, a complete and mature envelope protein, in its oligomericform, is preferable for inducing the formation of antibodies directedagainst conformation epitopes, among which are neutralizing antibodies(C. C. BRODER et al., Proc. Natl. Acad. Sci. USA, 1994, 91,11699-11703).

It has appeared that the elimination and replacement of the Cys residuesfrom the PID causes an envelope maturation defect which is no longerpresented in its mature form at the surface of the infected cells (SYUW. J., 1991, J. Virol., 65, 6349-6352, DEDERA et al., J. Virol., 1992,66, 1207-1209).

The two neighbouring cysteine residues of the peptide P237, conserved inthe ectodomain of the TM, in most retroviruses, define a loop structurewhich is the major constituent of the PID. The amino acid sequencebetween the two cysteine residues is highly conserved in the samespecies of lentivirus, such as FIV or HIV. On the other hand, when thevarious species of lentivirus, for example HIV-1 and FIV, are compared,although the two cysteine residues are conserved, the amino acidsequences situated between these two residues do not exhibit anyhomology (FIG. 1A).

As regards HIV, the Principal Immunodominant Domain (PID) of theenvelope of the human immunodeficiency type 1 virus (HIV-1) is highlyconserved among the various viral isolates of HIV-1. The sequencebetween the two cysteines of the PID is also conserved among the HIV-1subtypes, except for the 0 subtype (Outsider), which could correspond toa new type of HIV-1. The PID sequence of HIV-1 is completely differentfrom that of HIV-2, which is, on the other hand, identical to that ofthe simian immunodeficiency virus (SIV), to which HIV-2 isphylogenetically related.

In general, the PID of lentiviruses can cause the formation of viralinfection facilitating antibodies whose action has a deleterious effectopposite that of the protective antibodies generated by vaccination withthe Env protein, as specified above, for the FIV envelope protein.

Consequently, the applicant set himself the objective of providing Envproteins mutated at the level of the said principal immunodominantdomain (PID) of the transmembrane glycoprotein of lentiviruses andparticularly of the feline immunodeficiency virus or of the humanimmunodeficiency virus, which exhibit a significantly differentreactivity from that of the wild-type Env protein, particularly in thatthey do not induce the formation of deleterious, particularlyfacilitating, antibodies against the wild-type PID, while retaining thecapacity to produce a protective immune response, including inparticular neutralizing antibodies.

The subject of the present invention is, consequently, peptide fragmentscharacterized in that they constitute mutants of the wild-type principalimmunodominant domain (PID) of a wild-type transmembrane protein (TM) oflentiviruses and in particular of the feline immunodeficiency virus(FIV), the human immunodeficiency virus (HIV-1 and HIV-2) and thecaprine arthritis and encephalitis virus (CAEV), which peptide fragmentsare capable of modifying the immunogenic properties of the PID, suchthat the Env protein of lentiviruses containing them does not cause theproduction of facilitating or deleterious antibodies against thewild-type PID, whereas it retains the capacity to produce neutralizingantibodies.

Mutant of the PID is understood to mean, for the purposes of the presentinvention, at least one sequence between the two cysteines of awild-type PID of a TM protein of a lentivirus, which sequence ismodified (1) either in that at least one amino acid is mutated, (2) orin that it comprises the sequence of a PID of a lentivirus Env proteindifferent from that in which the said PID sequence is inserted. Forpractical reasons of insertion, the said peptide fragments may comprisedownstream and/or upstream of the said sequence between the twocysteines, at least one additional amino acid.

Such peptide fragments therefore replace the principal immunodominantdomain (PID) of a wild-type transmembrane protein (TM) of lentivirusesand in particular of the feline immunodeficiency virus (FIV), the humanimmunodeficiency virus (HIV-1 and HIV-2) and the caprine arthritis andencephalitis virus (CAEV) and constitute functional mutants of the PIDof the Env protein into which they are inserted.

Among the said fragments, there may be mentioned:

the fragment CNQNQWLCK, called f8, (SEQ ID No. 1),

the fragment CNQNQFLCK, called fd, (SEQ ID No. 2),

the fragment CNQNQLWCK, called fh, (SEQ ID No. 3),

the fragment CNQNQPFCK, called fm, (SEQ ID No. 4),

the fragment CEHQHFFCK, called n14, (SEQ ID No. 5),

the fragment CSMGTFFCK, called n19, (SEQ ID No. 6),

the fragment CLTDSFFCK, called n67, (SEQ ID No. 7),

the fragment CELKNFFCK, called n73, (SEQ ID No. 8),

the fragment CRPAAFFCK, called n92, (SEQ ID No. 9),

the fragment ELGCSGKLICKIP, called M5, (SEQ ID No. 10),

the fragment IWGCNQNQFFCTTAVPWN (SEQ ID No. 11),

the fragment IWGVAFRQVCTTAVPW (SEQ ID No. 12).

The sequences ID No. 1 to ID No. 10 are functional mutants of the FIVPID; in particular, the sequence ID No. 10 contains the sequence of theHIV-1 PID in an FIV sequence (that is to say in the context of FIV),whereas SEQ ID Nos. 11 and 12 are functional mutants of the HIV-1 PID:SEQ ID No. 11 contains the sequence of the FIV PID in the context ofHIV-1 (HIV-1/FIV chimera) and SEQ ID No. 12 contains the sequence of theSIV PID in the context of HIV-1 (HIV-1/SIV chimera). The sequences SEQID No. 1 to 9 are mutated sequences of the FIV PID.

The subject of the present invention is also Env proteins, characterizedin that they are mutated at the level of the principal immunodominantdomain (PID) of the transmembrane protein (TM) of lentiviruses and inparticular of the feline immunodeficiency virus (FIV), the humanimmunodeficiency virus (HIV-1 and HIV-2) and the caprine arthritis andencephalitis virus (CAEV), and in that they comprise one of thefragments as defined above, at the level of the PID, which proteins donot cause the production of deleterious antibodies, in particularfacilitating antibodies, against the wild-type PID, but conserveadequate conformation for the production of a protective immuneresponse, including, in particular the production of neutralizingantibodies for a protective vaccine application.

The subject of the present invention is also vectors for expressing Envproteins of lentiviruses, characterized in that they comprise at leastone mutated nucleic acid fragment encoding a mutated Env protein asdefined above.

Surprisingly, such vectors allow the expression of a functional viralenvelope, but whose immunological reactivity is significantly modified,in relation to that of the wild-type Env protein. Indeed, the mutatedand expressed protein does not cause the production of facilitatingantibodies against the wild-type PID, whereas it retains the capacity toproduce neutralizing antibodies.

According to an advantageous embodiment of the said expression vector,it consists of an expression vector comprising a sequence encoding anEnv protein of FIV, in which the sequence encoding PID (sequence2077-2115, with reference to the sequence of formula I of application EP0,577,458), is replaced by a sequence encoding any of the peptidefragments SEQ No. 1 to 10, as defined above.

The said vector may be advantageously constructed from a vector asdescribed in G. PANCINO et al., Virology, 1995, 206, 796-806.

According to another advantageous embodiment of the said expressionvector, it consists of an expression vector, comprising a sequenceencoding a HIV-1 Env protein in which the sequence encoding thewild-type PID is replaced by a sequence encoding a mutant of thewild-type principal immunodominant domain (PID) of a wild-typetransmembrane protein (TM), derived from the human immunodeficiencyvirus (HIV-1 and HIV-2) or of another lentivirus and capable ofmodifying the immunogenic properties of the PID; preferably, the saidsequence encodes either of the peptide fragments SEQ No. 11 or 12, asdefined above.

Preferably, the said sequences encoding PID are selected from thefollowing sequences (SEQ ID No. 13 to 24):

sequence encoding the sequence ID No. 1: TGT AAT CAA AAT CAA TGG CTT TGCAAA (SEQ ID No. 13), for the production of the vector pf8Δ20,

sequence encoding the sequence ID No. 2: TGT AAT CAA AAT CAA TTT TTA TGCAAA (SEQ ID No. 14), for the production of the vector pfdΔ20,

sequence encoding the sequence ID No. 3: TGT AAT CAA AAT CAA TTG TGG TGCAAA (SEQ ID No. 15), for the production of the vector pfhΔ20,

sequence encoding the sequence ID No. 4: TGT AAT CAA AAT CAA CCT TTT TGCAAA (SEQ ID No. 16), for the production of the vector pfmΔ20,

sequence encoding the sequence ID No. 5: TGT GAA CAT CAG CAT TTT TTC TGCAAA (SEQ ID No. 17), for the production of the vector pn14Δ20,

sequence encoding the sequence ID No. 6: TGT AGT ATG GGG ACG TTT TTC TGCAAA (SEQ ID No. 18), for the production of the vector pn19Δ20,

sequence encoding the sequence ID No. 7: TGT TTG ACA GAT TCG TTT TTC TGCAAA (SEQ ID No. 19), for the production of the vector pn67Δ20,

sequence encoding the sequence ID No. 8: TGT GAA CTC AAA AAC TTT TTC TGCAAA (SEQ ID No. 20), for the production of the vector pn73Δ20,

sequence encoding the sequence ID No. 9: TGT CGG CCA GCT GCT TTT TTC TGCAAA (SEQ ID No. 21), for the production of the vector pn92Δ20,

sequence encoding the sequence ID No. 10: GAG CTC GGA TGT TCT GGA AAACTC ATT TGC AAA ATC CCT (SEQ ID No. 22), for the production of thevector pM5Δ20,

sequence encoding the sequence ID No. 11: ATT TGG GGT TGC AAT CAA AATCAA TTC TTC TGC ACC ACT GCA GTG CCT TGG AAT-3' (SEQ ID No. 23),

sequence encoding the sequence ID No. 12: ATT TGG GGT TGC GCG TTT AGACAA GTC TGC ACC ACT GCA GTG CCT TGG AA-3' (SEQ ID No. 24).

The nucleic sequences according to the invention include all thesequences encoding the same amino acids (amino acid encoded by a codondifferent from that illustrated in sequences 13 to 24).

The subject of the present invention is also mutated lentivirusinfectious molecular clones, characterized in that they include asequence encoding a mutated Env protein, as defined above.

Preferably, the said mutated clones are obtained by introducing into aninfectious molecular clone of FIV, preferably into the infectiousmolecular clone p34TF10, a fragment containing the abovementionedmutations at the level of the sequences encoding PID, in place of thecorresponding wild-type sequences.

According to the mutated sequence introduced, the following clones areobtained: pTf8, pTfd, pTfh, pTn14, pTn19, pTn67, pTn73, pTn92 and pTM5.

Such clones are in particular obtained by incorporating into the genomeof FIV p34TF10 SpeI-BstBI 8287-8918 fragments of the said genome of FIVp34TF10 containing the abovementioned mutations, in place of thecorresponding wild-type sequences.

Also preferably, the said mutated clones are obtained by introducinginto an infectious molecular clone of HIV-1 a fragment containing theabovementioned mutations at the level of the sequences encoding PID, inplace of the corresponding wild-type sequences.

Such clones have the advantage of being able to be used directly as anattenuated live vaccine.

The subject of the present invention is also vaccine compositions,characterized in that they comprise at least one mutated Env proteinand/or at least one expression vector and/or at least one mutated clone,as defined above.

The subject of the present invention is, in addition, a process formonitoring an anti-lentivirus vaccination and/or for differentiatingbetween an anti-lentivirus vaccination and a lentivirus infection,characterized in that it comprises bringing a peptide fragment accordingto the invention into contact with a biological sample (blood inparticular) and detecting the antigen (peptide fragment)-antibody(produced during the vaccination or during the infection) complex formedby any appropriate means.

In such a process, the mutated PID serves advantageously as markersequence; indeed, the antibodies produced against the PID will bedifferent in the case of vaccination (mutated PID) and in the case ofinfection (wild-type PID). In such a context, if a viral infectionoccurs during the vaccination, it will be effectively possible todistinguish it, the two immunological responses being different, becauseof the presence of the mutated PID and of the absence of the wild-typePID, in the vaccinal composition.

It is possible, for the wild-type PID, to use detection systems such asthose described in European Patent Applications No. 0,564,477 of Nov.20, 1991, No. 0,577,458 of Jun. 16, 1993, and French Patent ApplicationNo. 94 07062 of Jun. 9, 1994; to detect the mutated PIDs, it is possibleto use similar detection systems, using the peptides of the mutated PIDas defined above, as reagents, instead of the wild-type PID (see Example1).

In addition to the above arrangements, the invention further comprisesother arrangements which will emerge from the description which follows,which refers to exemplary embodiments of the process which is thesubject of the present invention, as well as to the accompanyingdrawings, in which:

FIG. 1 represents the alignment of the sequences of the PIDs of the TMof various lentiviruses,

FIG. 2 represents the vector for expression of the envelope pTΔ20,

FIG. 3 represents the result of the fusion tests with the vectorcontaining the M5 sequence.

It should be understood, however, that these examples are given solelyby way of illustration of the subject of the invention and do notconstitute in any manner a limitation thereto.

EXAMPLE 1 Construction of Mutants in the FIV Context (Chimeras in whicha PID of HIV Replaces the Wild-Type PID of FIV, in an Env Sequence ofFIV)

The sequence encoding the Env protein of FIV is modified such that thesequences encoding the peptide fragments according to the invention asdefined above (SEQ ID No. 1 to 10) are inserted, in place of thesequence encoding the abovementioned peptide P237 (positions 2077-2115of the sequence encoding the Env protein as described in European PatentApplication No. 0,577,458); the said sequences encoding an Env proteinwhich are thus modified (mutant env sequences), are inserted into avector called pTΔ20, which comprises a deletion from nucleotide 923 tonucleotide 5410 in the FIV gene (gag and po1 genes).

The production of the vector pTΔ20 is described in G. PANCINO et al.,Virology, 1995, 206, 796-806.

The Env expression vector pTΔ20 (FIG. 2) is derived from the infectiousmolecular clone of FIV p34TF10, by deletion of the gag and po1 genes.

SpeI-BstBI 8287-8918 fragments of the env gene of FIV p34TF10,containing the abovementioned mutations are introduced into theexpression vector, in place of the corresponding fragments containingthe wild-type sequence.

To produce the env expression vector of FIV and to use it in felinecells, a deletion of the gag and po1 genes is made in the provirusp34TF10 (TALBOTT et al., Proc. Natl. Acad. Sci. USA, 1989, 86,5743-5747) by TthIII-EcoRV digestion and religation after treating withthe Klenow enzyme.

A clone, called pTΔ20, which comprises a larger deletion, fromnucleotide 923 to nucleotide 5410 effectively expresses the said Envprotein, after transfection into the CrFK cells (Virology, 1995, 206,796-806).

EXAMPLE 2 Mutated Peptides Obtained in Example 1 and Env ProteinsContaining Them: Functionality and Reactivity

Method:

To test the functionality of the mutated envelopes, the abovementionedexpression vectors (pf8Δ20, pfdΔ20, pfhΔ20, pfmΔ20, pn14Δ20, pn19Δ20,pn67Δ20, pn73Δ20, pn92Δ20, pM5Δ20,) producing mutated Env proteins inaccordance with the invention are transfected into CrFK cells andanalyzed for their capacity to induce the fusion of the cell membranes(formation of syncytia).

To carry out this test, it is necessary to transfect the said 10expression vectors, containing the mutated envelopes, into felinefibroblasts CrFK, in accordance with the following procedure:

A feline fibroblast cell line CrFK-H06T1/2 (OSBORNE et al., J. Gen.Virol., 1994, 75, 3641-3645) is cultured in a Dulbecco's modified Eaglemedium supplemented with 10% calf serum.

The plasmids are purified using the Promega maxiprep system and are thenextracted with phenol-chloroform. The transfection is carried out usingthe calcium phosphate precipitation method.

The transfection conditions are optimized and standardized usingplasmids expressing the β-galactosi-dase or luciferase reporter genes.

To test the reactivity of the mutated domains with the sera of catsinfected with FIV, an ELISA with the peptides according to the inventionis carried out. The microtitre plates, containing 96 wells (Immunolon®2), are coated with the said peptides, in a 0.1 M carbonate buffer pH9.6 (5 μg/ml), at 4° C. overnight.

After blocking with 3% bovine serum albumin, feline sera diluted in PBS,1% bovine serum albumin and 0.1% Tween 20 are added and incubated for 2hours.

After 5 washes with PBS, 1% Tween 20, anti-cat antibodies conjugatedwith peroxidase are incubated for 1 hour. After 5 washes, the reactionis developed using ABTS at 0.4 mg/ml and by reading the optical density30 min later.

In the same manner, the reactivity of the mutated domains can be testedwith human sera infected with HIV-1. In this case, the procedure is thesame, except for the use of 5% foetal calf serum in place of the bovineserum albumin.

Results:

Formation of syncytia:

The mutated vectors containing the sequences ID No. 1 to No. 10, producesyncytia, as illustrated in Table I below:

                                      TABLE I                                     __________________________________________________________________________                No. syncytia                                                                         No. syncytia                                                                         ELISA  ELISA                                           (No. nuclei) (No. nuclei) >25% TM2 >25% TM2                                  Peptides (0.5 μg) (2 μg) experiment natural                           __________________________________________________________________________    TM2 CNQNQFFCK                                                                             991 (5-100)                                                                          1900 (15-150)                                                f8 CNQNQWLCK 522 (5-100) 1280 (5-100) 1/8 1/11                                fd CNQNQFLCK ND°° 500 (5-90) 5/8 9/11                           fh CNQNQLWCK 200 (5-70) 415 (5-70 4/8 3/11                                    fm CNQNQPFCK 108 (5-50) 170 (5-50) 1/8 2/11                                   n14 CEHQHFFCK 187 (5-20) 754 (5-50) 0/8 2/11                                  n19 CSMGTFFCK 156 (5-30) 408 (5-80) 0/8 1/11                                  n67 CLTDSFFCK +* 800 (5-70) 0/8 0/11                                          n73 CELKNFFCK 160 (5-20) 700 (5-40) 0/8 1/11                                  n92 CRPAAFFCK 114 (5-30) 200 (5-30) NI.sup.# NI.sup.#                         0  3 (5-7) 4 (5-7)                                                          __________________________________________________________________________     *>> 100 syncytia/well                                                         °°ND: not determined                                            .sup.# NI: not interpretable in the ELISApeptide, because of the              background noise in the reaction of the peptide n92 with control sera of      noninfected cats.                                                        

The existence of cross-reactivity of the peptide n92 with the peptideTM2 was however ruled out by competition experiments between the twopeptides.

The mutated vector pM5Δ20, containing a mutated fragment of the HIV PIDwas tested in another experiment and also showed the formation ofsyncytia.

FIG. 3 illustrates the results obtained with the MS vector (comparedwith the wild-type W).

The chimeras HIV/FIV M5 are capable of inducing the formation ofsyncytia (FIG. 3) which are smaller in size (5-20 nuclei, 48 hours aftertransfection) than those induced by the wild-type (W).

The size and the number of syncytia induced by MS increases with time upto 72 hours.

Immunological reactivity:

The modifications produced in the immunological properties of the PIDsby the mutations which preserve the fusogenic capacity of the mutantenvelopes are tested by "peptide ELISA".

The peptides corresponding to the mutated PID (Table I) are synthesizedand used to coat ELISA micro-plates. 8 sera of cats, experimentallyinfected with 4 different isolates of FIV and 11 sera obtained fromnaturally infected cats are tested in order to evaluate the reactivityof these peptides.

The optical density values obtained with the mutant PID peptides arecompared with the values obtained with the wild-type PID peptide (TM2)comprising the peptide P237.

A substantial reduction in the reactivity of most of the mutants ishighlighted relative to the TM2 peptide.

Table I above shows, for each peptide, the number of sera which showed areactivity of more than 25% compared with the reactivity of the TM2peptide.

In some cases, with the sera which showed a reactivity with the mutantpeptides, competition assays were carried out between the TM2 peptideand the mutated peptides.

In no case does a mutated peptide inhibit the reactivity of the serumtowards the TM2 peptide, whereas the TM2 peptide completely inhibits thereactivity of the sera of cats infected with the mutated proteins.

These results show that the mutations introduced into the FIV PIDgreatly modify its recognition by sera of cats infected with an FIV(significantly modified immunoreactivity).

Cross-reactivity between the PIDs of FIV and HIV-1:

Two ELISAs were developed to test either the reactivity of the FIV PIDwith human serum infected with HIV-1, or the reactivity of the HIV-1 PIDwith cat serum infected with FIV.

The reactivity of the sera of 8 patients infected with HIV-1 and of 10cats naturally infected with FIV, with the peptides corresponding to thePIDs either of HIV-1 or FIV, was tested with an ELISA.

The peptides p237 and SP89029 (ANRS Catalogue, 1994) corresponding tothe FIV and HIV-1 sequences (PID) respectively and 2 peptides chemicallycyclized between the two cysteines (Y-15-Vc (YQELGCN NOFFCLV) andL-15-Tc (LLGIWGCSGKLICTT) respectively for FIV and HIV-1), so as tomimic the natural folding, were used to coat the microtitre plates.

The sera of individuals infected with HIV-1 react strongly with thepeptides containing a PID derived from HIV-1 (SP89029 and L-15-Tc), butnot with the FIV peptides, that is to say the peptides containing a PIDderived from FIV (P237 and Y-15-Vc) and, correspondingly, the sera ofcats infected with FIV recognize the FIV peptides, but not the HIV-1peptides.

Thus, no cross-reaction can be detected between the two PIDs. Thisallows the use of an appropriate peptide ELISA to monitor the anti-HIV-1vaccination procedures based on chimeric envelopes containing the FIVPID sequence in the HIV-1 context or other mutations which modify theantigenic properties of the HIV-1 PID.

A similar approach is also possible in the case of vaccination againstFIV, using an HIV-1 PID or other mutated sequences of the FIV envelope.

EXAMPLE 3 Gene Vaccination

The administration of the FIV env gene products as defined above, byinjection of crude DNA, was studied so as to allow a vaccinal procedurewhich would allow analysis of the immune response of the wild-typeenvelope proteins, compared with the envelopes mutated in the PIDregion.

The expression vector Env pTΔ20, derived from the infectious molecularclone of FIV p34TF10, by deletion of the gag and po1 genes, is used tovaccinate cats.

The vector pTΔ20 is injected intramuscularly into 4 cats (4 injectionsof 200-400 μg of DNA). The antibody response is analysed using 4different peptides corresponding to the linear B cell epitopes (SU2,SU3, TM2 and TM4) by ELISA and by immunoprecipitation of the Envglycoproteins, from lysates of a cell line FL4, infected with FIV, after³⁵ S labelling.

3 of the 4 cats show a weak response to at least 2 peptides. One serumis also capable of precipitating the envelope glycoproteins (lastdilution tested 1:320) from the FL4 cells. These results indicate thatthe immunization based on the env gene indeed leads to an immuneresponse and therefore to its use in vaccines with the mutated Envproteins.

EXAMPLE 4 Construction of Mutants in the HIV-1 Context (Chimeras inwhich a PID of FIV or SIV Replaces the Wild-Type PID of HIV-1, in anHIV-1 Env Sequence): Functionality and Reactivity of the ProductsObtained

The sequence encoding the HIV-1 Env protein is modified so that thesequences encoding the peptide fragments SEQ ID No. 11 or 12, as definedabove, are inserted in place of the sequence encoding the peptidefragment contained between the two cysteines of the HIV-1 PID.

Sequences encoding modified Env proteins are thus obtained in which thesequence contained between the two cysteines of the HIV-1 PID, in theHIV-1 LAI envelope, has been replaced by the corresponding sequences ofthe feline immunodeficiency virus (FIV) or of SIV.

To do this, the SmaI-BamHI fragment of the HIV-1 LAI envelope wassubcloned into the plasmid pTZ18 and the mutations were introduced bysite-directed mutagenesis (Kunkel), using oligonucleotides correspondingto the mutations, as specified above:

    __________________________________________________________________________    P   s  t  F  I   V  ;                                                            - 5'-ATTTGGGGTTGCAATCAAAATCAATTCTTCTGCACCACTGCAGTGCCTTGGAAT-3';              - P  s  t  S I V  ;                                                           - 5'-ATTTGGGGTTGCGCGTTTAGACAAGTCTGCACCACTGCAGTGCCTTGGAA-3',                 __________________________________________________________________________

To allow screening of the recombinant clones, a Pst1 restriction sitewas also introduced into the envelope upstream of the PID, so as not tomodify the amino acid sequence. The mutated fragment was then clonedinto the expression vector pMA243 (DRAGIC T. et al., J. Virol., 1992,66, 4794-4802), in order to analyse the properties of the mutantenvelopes.

The capacity of the mutant envelopes to induce fusion between cellmembranes, a property of the wild-type envelope, was analysed using afusion test carried out after transfection of the envelopes into COScells and coculture with HeLa cells expressing the HIV-1 cellularreceptor CD4 and the β-galactosidase gene placed under the control ofthe HIV-1 LTR (DRAGIC T. et al., cited above; CHARNEAU P. et al., J.Virol., 1992, 66, 2814-2820). The fusion of two cell types, mediated bythe HIV-1 envelope, activates the expression of β-galactosidase and thefused cells can be detected by a blue colour, derived from an enzymaticreaction. After transfection of 3 μg of DNA/well into 6-well plates, thetwo mutant envelopes are induced by cellular fusions and the formationof syncytia (Table II). In particular, the chimeric clone containing theFIV sequence in the context of the HIV-1 envelope showed good efficiencyin inducing cell fusion.

                  TABLE II                                                        ______________________________________                                        Clone            No. of blue foci/well*                                       ______________________________________                                        WT (wild-type HIV-1 LAI)                                                                       3500                                                           HIV-1/FIV 1400                                                                HIV-1/SIV  200                                                              ______________________________________                                         *mean of the count for 3 wells                                           

The expression and maturation of the mutant envelopes were analysed bymetabolic labelling with [³⁵ ]S methionine and cysteine of thetransfected cells, followed by immunoprecipitation of the envelopeproteins with a pool of human sera obtained from seropositive donors.This study showed that the envelope precursors of the two mutants wereproduced in a quantity comparable to the wild-type clone. The precursorsof the two mutant envelopes were correctly cleaved to give rise to twosubunits of the mature complex of the envelope, the extracellularglycoprotein (SU) and the transmembrane glycoprotein (TM). Theimmunoprecipitation of the SU glycoproteins of the culture supernatantsshowed that the SUs of the mutated envelopes were liberated into thesupernatant in a quantity greater than that of the wild-type envelope.

As evident from the above, the invention is not at all limited to itsembodiments, implementations and applications which have just beendescribed more explicitly; it embraces on the contrary all the variantsthereof which may occur to a specialist in this field, without departingfrom the scope or from the framework of the present invention.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 24                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Cys Asn Gln Asn Gln Trp Leu Cys Lys                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Cys Asn Gln Asn Gln Phe Leu Cys Lys                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Cys Asn Gln Asn Gln Leu Trp Cys Lys                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Cys Asn Gln Asn Gln Pro Phe Cys Lys                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Cys Glu His Gln His Phe Phe Cys Lys                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Cys Ser Met Gly Thr Phe Phe Cys Lys                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Cys Leu Thr Asp Ser Phe Phe Cys Lys                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Cys Glu Leu Lys Asn Phe Phe Cys Lys                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Cys Arg Pro Ala Ala Phe Phe Cys Lys                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Glu Leu Gly Cys Ser Gly Lys Leu Ile Cys Ly - #s Ile Pro                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Ile Trp Gly Cys Asn Gln Asn Gln Phe Phe Cy - #s Thr Thr Ala Val        Pro                                                                             1               5   - #                10  - #                15              - - Trp Asn                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Ile Trp Gly Cys Ala Phe Arg Gln Val Cys Th - #r Thr Ala Val Pro Trp      1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "OLIGONUCLEOTIDE"                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - TGTAATCAAA ATCAATGGCT TTGCAAA          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "OLIGONUCLEOTIDE"                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - TGTAATCAAA ATCAATTTTT ATGCAAA          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "OLIGONUCLEOTIDE"                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - TGTAATCAAA ATCAATTGTG GTGCAAA          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "OLIGONUCLEOTIDE"                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - TGTAATCAAA ATCAACCTTT TTGCAAA          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "OLIGONUCLEOTIDE"                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - TGTGAACATC AGCATTTTTT CTGCAAA          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "OLIGONUCLEOTIDE"                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - TGTAGTATGG GGACGTTTTT CTGCAAA          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - TGTTTGACAG ATTCGTTTTT CTGCAAA          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "OLIGONUCLEOTIDE"                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - TGTGAACTCA AAAACTTTTT CTGCAAA          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "OLIGONUCLEOTIDE"                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - TGTCGGCCAG CTGCTTTTTT CTGCAAA          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "OLIGONUCLEOTIDE"                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - GAGCTCGGAT GTTCTGGAAA ACTCATTTGC AAAATCCCT      - #                      - #    39                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "OLIGONUCLEOTIDE"                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - ATTTGGGGTT GCAATCAAAA TCAATTCTTC TGCACCACTG CAGTGCCTTG GA - #AT               54                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "OLIGONUCLEOTIDE"                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - ATTTGGGGTT GCGCGTTTAG ACAAGTCTGC ACCACTGCAG TGCCTTGGAA  - #                  50                                                                       __________________________________________________________________________

We claim:
 1. A purified mutant lentivirus Env protein comprising apolypeptide having at least one mutated amino acid sequence of atransmembrane protein of a principal immunodominant domain (PID),wherein said amino acid sequence is mutated between two cysteines. 2.The purified mutant lentivirus Env protein according to claim 1, whereinsaid lentivirus is selected from the group consisting of the felineimmunodeficiency virus (FIV), the human immunodeficiency virus (HIV),and the caprine arthritis and encephalitis virus (CAEV).
 3. An isolatedpolynucleotide comprising a nucleotide sequence encoding the mutant Envprotein according to claim
 1. 4. The polynucleotide according to claim 3containing a sequence selected from the group consisting of SEQ ID No.13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ IDNo. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQID No. 23, and SEQ ID No.
 24. 5. The isolated polynucleotide accordingto claim 3, wherein said nucleotide sequence encodes a mutant FIV Envprotein.
 6. The isolated polynucleotide according to claim 3, whereinsaid nucleotide sequence encodes a mutant HIV Env protein.
 7. Theisolated polynucleotide according to claim 3, wherein said nucleotidesequence encodes a mutant CAEV Env protein.
 8. An expression vectorcomprising the polynucleotide of claim
 3. 9. The expression vectoraccording to claim 8, wherein said polynucleotide encodes a modifiedPID.
 10. The expression vector according to claim 9, wherein saidencoded modified PID is selected from the group consisting of SEQ ID No.1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6,SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11,and SEQ ID No.
 12. 11. A process of producing an antibody comprising thesteps of:(a) administering the polypeptide of claim 1 to a host toinduce formation of said antibody; and (b) isolating the antibody fromthe host.
 12. An immunogenic composition comprising at least oneexpression vector according to claim
 8. 13. An immunogenic compositioncomprising at least one expression vector according to claim
 9. 14. Animmunogenic composition comprising at least one expression vectoraccording to claim
 10. 15. An immunogenic composition comprising atleast one mutated Env protein according to claim
 1. 16. Alentivirus-specific diagnostic reagent, comprising a polypeptidesequence according to claim 2.